Every year, pests detrimental to agriculture, forestry, and public health cause losses in the millions dollars. Various strategies have been used control such pests.
One strategy is the use of chemical pesticides with a broad range or spectrum of activity. However, there are a number of disadvantages to using such chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. An example of a biopesticide is a bacterium which produces a substance toxic to the infesting pest. A biopesticide is generally less harmful to non-target organisms and the environment as a whole than chemical pesticides.
The most widely used biopesticide is Bacillus thuringiensis. Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and insect-rich environments. During sporulation, Bacillus thuringiensis produces a parasporal crystal inclusion(s) which is insecticidal upon ingestion to susceptible insect larvae of the order Lepidoptera, Diptera, or Coleoptera. The inclusion(s) may vary in shape, number, and composition. They are comprised of one or more proteins called crystal delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal crystal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255) .
There are several Bacillus thuringiensis strains that are widely used as biopesticides in the forestry, agricultural, and public health areas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai have been found to produce crystal delta-endotoxins specific for Lepidoptera. Bacillus thuringiensis subsp. israelensis has been found to produce crystal delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112). Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203), has been found to produce a crystal delta-endotoxin specific for Coleoptera. Several Bacillus thuringiensis crystal delta-endotoxin proteins are also reportedly pesticidal to nematodes, Acari, Hymenoptera, Phthiraptera, Platyhelminthes, Homoptera, Blattodea, and Protozoa.
The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Herrnstadt et al., 1986, Bio/Technology 4:305-308; Herrnstadt and Soares, 1988, U.S. Pat. No. 4,764,372). This strain, designated "Bacillus thuringiensis subsp. san diego", M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number NRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publicly acknowledged that Bacillus thuringiensis subsp. san diego is Bacillus thuringiensis subsp. tenebrionis. Furthermore, the '372 patent has been assigned to Novo Nordisk A/S. A spo.sup.+ cry.sup.- (asporogenous crystal forming) mutant of M-7 has purportedly been obtained by culturing M-7 in the presence of ethidium bromide (Herrnstadt and Gaertner, 1987, EP Application No. 228,228). However, there was no indication of increased production of delta-endotoxin, increased parasporal crystal size, and/or increased pesticidal activity relative to the parental, M-7 strain.
The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The six major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), Diptera-specific (cryIV) (Hofte and Whiteley, 1989, Microbiol. Rev. 53:242-255), Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275).
The utility of Bacillus thuringiensis strains for the control of pests is dependent upon efficient and economical production of the active toxins. This in turn is dependent upon the amount of crystal delta-endotoxins which can be produced by fermentation of the active Bacillus thuringiensis strains.
Consequently a recognized need for products of improved strength exists.
One way to fulfill this need would be to concentrate the preparations. However, this would add considerably to the production cost in comparison to the savings obtained in storage and transportation.
A much more elegant solution would be to create mutants of existing B.t. strains which produce substantially larger amounts of delta-endotoxin and have a substantially higher amount of pesticidal activity compared to its parental strain. Such mutants would give a more efficient and economical production of active delta-endotoxins and a possibility for manufacture of B.t. products with increased potency at equal or lower cost. This in turn would be an advantage for the user as reduced volumes of pesticide formulation have to be stored and handled for a given acreage. In addition, the users will have less container material to dispose of, thereby reducing the impact on the environment.